1 research outputs found
Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection
Chemical
analysis of small extracellular vesicles (sEVs) circulating
in body fluids holds potentials in noninvasive diagnosis of diseases
and evaluation of therapeutic treatments. However, quantification
of sEVs remains a challenge due to lacking of cost-effective analytical
protocols. Herein we report a facile method based on size exclusion
chromatography with fluorescence detection (SEC-FD) for sEVs quantification.
After removal of cells and cell debris, a 0.50 mL sample (e.g., cell
culture medium) is incubated with CM-Dil dye to fluorescently label
sEVs. The incubation solution is then separated on a SEC column packed
with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553
nm/Em570 nm by using a fluorometer equipped with a 50-μL flow
through cuvette. Separation efficiency of the proposed SEC-FD method
was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate.
Liposomes were eluted out in less than 6 min, about 10 min before
albumin-FITC. A separation repeatability (RSD in retention time) of
1.4% (<i>n</i> = 5) was obtained for liposomes. In analysis
of cell culture media, linear calibration curves based on SEC-FD peak
height versus sEVs concentration were obtained with <i>r</i><sup>2</sup> value of 0.996. Intraday quantification repeatability
(RSD in peak height) was 3.2% (<i>n</i> = 5). The detection
limit was estimated to be 2.9 × 10<sup>7</sup> exosome particles/mL.
The proposed assay was applied to the first study of sEVs secretion
from TK6 cells cultured in serum-free medium for a culturing period
from 1 to 48 h